The translational control of protein synthesis by hemin in rabbit reticulocytes is mediated by the formation of a high molecular weight protein inhibitor of polypeptide chain initiation termed the hemin-controlled translational repressor (HCR). HCR acts as a specific protein kinase by phosphorylating the initiation factor (eIF-2) that mediates binding of the initiator tRNA (met-tRNAf) to 40 s ribosomal subunits. We plan to determine the precise molecular effect (s) the phosphorylation of eIF-2 has on polypeptide chain initiation. The effect of HCR on polypeptide chain initiation will be studied in the intact, unfractionated lysate using such purified and radiolabelled probes as globin mRNA, Met-tRNAf, eIF-2, and eIF-3 to measure initiation complex formation. The role that deacylation of subunit-bound Met-tRNAf plays in the regulation by HCR will be explored, and the ribosomal Met-tRNAf hydrolase that appears to mediate this effect will be purified and characterized. Finally, the alpha subunit of eIF-2, phosphorylated by HCR, will be isolated and subjected to peptide analysis to determine whether phosphorylation occurs at one or multiple sites.